The goal of this project is to characterize structure-function relationships for the multifunctional flavoprotein, PutA from Escherichia coli. This remarkable protein is both a transcriptional repressor of the proline utilization (put) regulon and a membrane-associated proline catabolic enzyme. The three-dimensional structural basis for the versatility of PutA is unknown. The working hypothesis whereby PutA changes its intracellular location and function is that conformational changes governed by the flavin redox state control its macromolecular associations (i.e. DNA and membrane-binding). The proposed research addresses three fundamental outstanding questions related to PutA structure and function: (1) What is the three-dimensional structure of PutA? (2) How does PutA interact with DNA? and (3) What are the conformational changes that allow PutA to function as both a DNA-binding protein and a membrane bound enzyme? The first aim of this proposal is to determine the three-dimensional structure of PutA using X-ray crystallography. Crystallization of PutA is challenging due to its large size (1320 amino acid residues) therefore a "divide and conquer" strategy will be employed in which shorter polypeptides that retain one or more of the functions of PutA will be engineered and crystallized separately. These smaller structures will then be stitched together computationally to derive a model of the full-length protein. Good progress has already been made using this approach - the 2.0 A crystal structure of a protein corresponding to the first 669 residues of PutA has been solved. The second aim is to determine the structural basis for PutA-DNA interactions by solving the crystal structures of PutA and truncated PutA proteins complexed with well-defined DNA binding sites. The third aim is to explore the conformational changes induced by proline reduction of the flavin by determining the crystal structures of PutA and truncated PutA proteins in the proline-reduced state. These studies will contribute pivotal understanding into the regulatory mechanism of PutA and timely knowledge of its structure.